ISHS Acta Horticulturae 808: II International Symposium on Tomato Diseases
SPECIFICALLY HRPL GENE PRIMERS FOR DETECTING OF PSEUDOMONAS SYRINGAE PV. TOMATO BY POLYMERASE CHAIN REACTION |
| Authors: | A. Abdelmonem, M.R. Rasmy |
| Abstract:
The detection of Pseudomonas syringae pv. Tomato (PST), the causative of bacterial speck of tomato, was performed on tomato leaves and fruits by polymerase chain reaction amplification of a specific DNA fragment of the hrpL sequence. The consensus primers hrpL1 and hrpL2 were designed based on the alignment of pseudomonad hrpL gene sequences available in nucleic acid data banks. This primer set produced a 631-bp amplicon from 8 of the 15 pseudomonads isolates tested. These isolates belonged to genomospecies 1 and 2. The amplicon obtained from 8 of these isolates was digested with eight restriction enzymes. Three different restriction patterns were produced from isolates belonging to genomospecies 1, resulting in A1 and A2 patterns, while isolates belonging to genomospecies 2 were characterized by a B pattern. Patterns A1 and A2 differed at only two sites, a Bsp 143I site located at nucleotide 360 and a MseI site located at nucleotides 22– 24. Group A2 consisted solely of P. syringae pv. tomato isolates . The hrpL gene in P. syringae pv. tomato isolates was sequenced. Two primer sets, tom1/tom2 and tom1/tom3, were designed and tested for specificity to P. syringae pv. tomato. These primers amplified expected fragments of 242 and 303 bp, respectively. tom1/tom2 amplified a fragment only with P. syringae pv. tomato DNA, while tom1/tom3 amplified all tested strains belonging to genomospecies 1. A diagnostic procedure using the Tom1/Tom2 primer set was success for the detection of P. syringae pv. tomato in diseased fruit and artificially inoculated leaves. The DIA and ELISA techniques were the least sensitive, requiring populations of 106mL–1 and 105mL–1, respectively, for positive detection of (PST). | |
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